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Formation and Production of In Vitro Meat
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This entry is adapted from the peer-reviewed paper 10.3390/su14094910

In vitro meat (IVM) is a type of meat that is produced using animal cells, under laboratory conditions. IVM has received global attention and is considered a meat of the future by many advocates. This is because IVM production is more sustainable and less emission intensive than the meat obtained from traditional farming. IVM production will also reduce animal welfare issues such as production of factory farmed animals and brutal slaughtering of animals that are associated with traditional farming. IVM can confer direct benefits to public health, such as reducing the spread of food-borne illness. Despite the environmental and public health benefits, commercialisation of this technology is still facing multiple hurdles. The most significant challenges for IVM are its price, consumer perception, and acceptance of technology used to produce IVM.

in vitro meat IVM production self-organising technique

1. General Background of IVM Muscle Formation

IVM works on the concept of myogenesis, a process of muscle formation. The formation of muscular tissue occurs during embryonic development, by multipotent myoblasts cells of mesodermal origin. These myoblast cells undergo embryonic fusion, proliferation and differentiation [1][2], to form myotubes, which gradually combine to forms muscle fibres. The process of skeletal muscle formation is analogous to the production of IVM because a piece of meat is merely a mass of edible muscle fibres. It was published by Buckingham et.al in 2003 [3] has more detailed information on the process of muscle formation [4].

1.1. IVM History and Development

IVM is also known as ‘cultured meat’, ‘lab-grown meat’ and ‘clean meat’ [5][6]. It has also been referred to as ‘animal-free meat’, ‘slaughter-free meat’, ‘vat meat’ ‘synthetic meat’, ‘artificial meat’, ‘shmeat’, ‘frankenmeat’ and ‘test-tube meat’. The Food and Drug Administration (FDA, Silver Spring, MD, USA), United States Drug Administration (USDA, Silver Spring, MD, USA) and other in vitro meat companies have jointly termed IVM as ‘cell-based meat’ [7] The most popular IVM term used is ‘cultivated meat’.
The concept of IVM has been prevalent since Winston Churchill in 1931 predicted the future and stated that, “fifty years hence, it shall escape the absurdity of growing a whole chicken in order to eat the breast or wing, by growing these parts separately under a suitable medium” [8]. Research on in vitro cultivation of muscle fibres only began as early as 1971 when a researcher cultured immature aortal cells from guinea pig to obtain myofibrils on eight weeks of culturing [9]. A few years later, another researcher cultured goldfish cells, which eventually developed into fish fillets [10]. During this period, a Dutch researcher turned entrepreneur, Willem Van Eelen received a patent for producing edible meat using collagen and muscle cells without killing animals [11]. In 2014, People for the Ethical Treatment of Animals (PETA), a US-based nonprofit organization, exhibited their support towards IVM by announcing a prize money of 1 million dollars to those who could cultivate lab-grown meat/in vitro meat (IVM) using chicken cells [12]. Besides that, the National Aeronautics and Space Administration (NASA) supported the idea of IVM since it can be used as a long-term food product for space missions [13]. IVM regained popularity as Dr Mark Post in 2013 created a beef burger patty using cow cells in a live event in London (BBC, 2013). Following this event, production of cell-based meat using IVM technology has created much attention in the past few years and is currently one of the most researched topics in food science.
Currently, IVM research is supported by nonprofit organisations such as the Good Food Institute (GFI), and start-up companies such as New Harvest, CUBIQ Foods, Mosa Meat, Memphis Meats and JUST Inc. Furthermore, business tycoons such as Bill Gates, Sergey Brin and Richard Branson have invested in cell-based meat research and IVM start-up companies [14]. Hitherto, the number of investors in IVM and sustainable foods has increased exponentially. These investments have had a significant impact on the production of IVM as the costs for an IVM burger patty is now about $2400, which was $330,000 previously [15]. However, in 2018, IVM proponents hoped that the price will be as low as $5 by 2021 [16]. According to the Forbes website March 2022, the price of cell-cultured meat has decreased from $330,000 to about €9 or $9.80 per burger, which is now much more affordable.
The IVM industry is ever-increasing, and many start-up companies in the US and European countries now produce cell-based meats. In addition, even southeast Asian countries have extended their support towards IVM with China recently signing a 300-million-dollar deal to import slaughter-free meat from an Israel firm [17]. Japan has also struck a deal with JUST Inc. to deliver cell-based Wagyu beef in the market [18]. According to industry experts, IVM will be commercially available in about five years due to huge technological advancement.

1.1.1. Negative Repercussions of Traditional Meat Industry and the Need for Meat Alternatives

The Food and Agriculture Organisation (FAO) predicted that the rate of meat consumption will increase by two thirds, for example, about 73% in 2050 [19]. Such a tendency is expected to last for nearly four decades [20]. In other words, an increase in meat consumption levels will result in increased demand for meat products. This increase in meat production will result in increased adverse effects such as global climate changes, global warming, greenhouse gas emissions (GEG), increased carbon footprint and animal suffering. Besides, traditional meat production also causes water pollution due to the increased use of fertilisers and pesticides [21]. According to the United Nations (UN) and FAO, about 30% land usage, 6% of freshwater usage and 14.5% of greenhouse gas emissions are linked to the livestock meat industry [20][22]. Moreover, New Zealand alone emits 49.2% of its total GHG emissions from the agricultural sector [23], which includes both the livestock and meat industry.
For all the reasons mentioned above, there is a dire need across the world for sustainable meat alternatives such as IVM to meet the global demand for meat. According to few researchers, IVM takes up 99% less land, 45% less energy, 96% fewer greenhouse gases emissions compared to the traditional meat industry [24][25][26][27]. The greenhouse gases (GHG) emissions of IVM are as low as traditional pork and poultry industries [27][28]. Furthermore, IVM is unlikely to cause any adverse environmental impacts and may in fact promote the reversal of climate changes [29].

2. IVM Production

2.1. Cells and Culture Conditions

2.1.1. Cells

There are various cell sources for IVM production, and these include adult stem cells (ASC), tissue-specific stem cells, mesenchymal stem cells (MSC), and induced pluripotent stem cells (iPSCs). The most widely used cells are embryonic stem cells and myosatellite cells [30][31].

Myosatellite Cells

Satellite cells are the most preferred cell sources to produce IVM skeletal muscle tissue. Satellite cells are also known as myoblast cells, myosatellite cells or muscle stem cells, which belong to the adult stem cells (ASC) category. It exhibits multipotency and is analogous to embryonic myoblast cells [7]. Satellite cells are mononucleated adult stem cells (ASC) situated at the periphery of skeletal muscle myofiber. When the myofibers are damaged in an injury, the satellite cells become activated, divide, and fuse to replace the damaged myofibers. These myofibers eventually form myofibrils and muscle tissues at later stages. Similar effects will be observed if these cells are used in IVM production. Hence satellite cells are the most preferred cell source.
It was supported that satellite, myoblast, or adult stem cells are the most suitable type of cells due to their high regeneration power [32][33], ability to replicate myogenesis [30][34], and capacity to produce mature cells with specialised morphologies and functions, unlike the embryonic stem cells (ESCs). Hence, these cells are widely used in IVM research [35], especially the C2C12 myoblast cells [36][37][38][39][40][41], and in bio-artificial muscle production (BAMS) [42][43]. Moreover, adult stem cells (ASCs) such as epithelial cells have been used previously in the in vitro muscle production system [31]. However these cells require stimulation to form myoblasts, which may result in faulty myogenesis [44]. Besides that, myosatellite, myoblast or ASC’s have a few drawbacks such as limited proliferation rate and susceptibility to turning cancerous, if cultured for extended periods.

Embryonic Stem Cells (ESC)

Stem cells that are derived from the embryo are known as embryonic stem cells (ESC) [45], which are either extracted from bovine or porcine. These are preferred cell sources for IVM production [46] due to their pluripotency and unlimited self-renewal capacity. For this reason, some researchers claim that IVM produced using ESC could provide sufficient meat to feed global hunger [47]. Thus, for the above reasons, ESC is considered a potentially good cell source for IVM production. However, there is currently no availability of bovine, ovine or porcine-derived ESC, with only murine cell lineages available. Hence ESC has to be differentiated into myogenic progenitor cells (MPCs) before muscle fibres can be formed. Besides that, other concerns of ESC in IVM production are its requirements of cell differentiation to produce myoblasts [44][47][48]. These stimulated cells are susceptible to loss of their existing proliferative characteristics at any later stages, despite culturing [49]. In addition, there is difficulty in maintaining undifferentiated embryonic stem cells [50][51], as opposed to other cell sources for IVM production.

Induced Pluripotent Stem Cells (iPSC’s)

Induced pluripotent stem cells are differentiated cells, which are already transfected to induce pluripotency in cells. These iPSC’s can be used as a cell source for IVM production [7][52] due to their myogenic differentiation capacity and injury repair mechanism [53]. However, there is no scientific evidence regarding their usage in IVM production. Only one used fibroblast cell co-cultured with goldfish explant in the production of a bio-artificial muscle system (BAMs) [54]. On a different note, iPSC’s are often co-cultured with fat cells such as adipose tissue-derived stem cell (ADC) to improve the texture, flavour and tenderness of IVM by increasing the amount of intramuscular fat [55]. These co-culturing techniques have been observed in others  [38][56][57].

Dedifferentiated Cells

Dedifferentiated cells are the cells that have been reversed from terminally differentiated cells into multipotent cells, such as mature adipocytes. These cells on dedifferentiation give rise to multipotent preadipocyte cell line known as dedifferentiated fat cells (DFAT) [57]. In addition, these cells produce skeletal myocytes (muscle cells) when transdifferentiated [38]. Dedifferentiated cell properties make it suitable as a cell source for IVM production. However, some researchers argue that terminally differentiated cell properties of transdifferentiation, dedifferentiation and multipotency may be unusual features exhibited by cell-like substances, rather than the cell [36][58][59].
Despite the variety of cell sources available, IVM production is still a challenging task due to chances of cell death. However, some were suggested that death can be prevented either by using immortal cells or by immortalisation of cell lineage. Furthermore, another challenge is to create an environment (in vitro) that mimics in vivo conditions for the optimum growth of cells. Detailed information on culture conditions is covered in Section 5.1.2.

2.1.2. Culture Conditions

Typically, culture conditions include factors such as culture media, serums, growth hormones and parameters such as pH, temperature, oxygen potential, pressure, and mechanical/electromagnetic/gravitational simulations for cells to produce IVM. However, defining a range, as well as optimising and stabilising the above parameters, is a challenging task, during large scale IVM production.

Culture Medium

The culture medium plays a vital role in IVM production because it serves as a nutrient source for the growth of cells. The culture medium must be simple, edible optimal, affordable, and readily absorbable because it is used in considerable amounts. Traditionally, natural medium such as blood plasma was used for animal cell culture [60], but currently, researchers work with inexpensive serum-free medium such as Dulbecco’s Modified Eagle Medium (DMEM) [30][36][37][43][61] and Ultroser G, which has all the essential nutrients, growth factors, adhesion factors and binding proteins [30]. However, the disadvantage of an animal-friendly and serum-free medium is the cost involved. Nevertheless, few researchers suggest that culture medium with both plant-based extracts and partially purified growth factors are economical and beneficial for the growth of cells [37]. Besides, the requirement for culture medium changes depending on cell growth or stages of development, such as differentiation and proliferation media used. Thus, there is a growing need for a continuous supply of edible and animal-friendly medium, which facilitates the growth of cells.

Serum

Traditionally, animal-based serums such as foetal bovine serum (FBS) [58][62][63], foetal calf serum (FCS) [7], fishmeal extract and horse serums were used. However, these serums are neither ethical nor economical to use in the long run. Furthermore, animal-based serums have a high risk of carrying pathogens [45]. Researchers are now working with animal-free serum such as algal (cyanobacteria) or fungal/mushroom-based (Shiitake and Maitake mushrooms) [64], which makes it animal-friendly in nature. Interestingly, these serum-free medium have higher growth rates compared to FBS [44], due to sphingosine 1-phosphate and amino-acids in mushrooms [36][45].

Growth Factors

Growth factors are essential components for the production of IVM [65], but the formulation and optimisation of growth factors is a challenging task [66] because it is dependent on the type of cells used. The commonly used growth factors for IVM production include Transforming Growth Factor-β (TGF-β), Fibroblast Growth Factors (FGFs), Insulin-like Growth Factors (IGF) VEGF (vascular endothelial growth factor), and FGF2 (fibroblast growth factor) [67]. However, TGF-β decreases the myoblast recruitment and differentiation [68], whereas FGFs are more stimulatory in nature compared to TGF—β. FGFs can enhance the rate of myoblast proliferation and prevent differentiation [69]. Similar to FGFs, a splice variant of IGF-1, called the mechano growth factor, increases proliferation of myoblasts [70] and differentiation in C2C12 myoblasts [71].

2.2. Processes Involved in IVM Production

2.2.1. Self-Organising Technique

The self-organising technique is a scaffoldless tissue engineering technique, which allows cells or tissues to grow freely using external forces such as physical manipulation or thermal input. Additionally, self-organisation works on the principle of tissue fusion. Tissue fusion is a process in the developmental biology stage, where two or more identical tissues meet and fuse to form a continuous structure [72][73]. The tissues produced using the self-organising technique have native tissue morphology and can be grown up to several centimetres [7][39][74].
The self-organising technique was first reported by Benjaminson et al. (2002), who produced the first-ever in vitro meat using goldfish (Carassius auratus) skeletal muscle explant, which regenerated and rearranged itself without any matrix, and showed a growth rate of 79% from the initial state. The explant was identical to the goldfish skeletal muscle and resembled fish fillets in terms of appearance and odour [75].
The self-organising technique is beneficial as it mimics skeletal muscles by retaining all the tissues which form the meat. Another benefit is its ability to produce a highly structured meat, unlike other methods. However, there are significant drawbacks with this method such as its susceptibility to undergo necrosis in the absence of blood supply [76]. Furthermore, IVM produced by this method requires the need for in vivo blood supply or vascularisation and an excretory mechanism to expel metabolic waste [31]. A major drawback of the self-organising technique is its inability to produce highly structured meat because it produces nonstructured and soft-consistency meat, which is only suitable for sausages, minced meat and burger patties [30].

2.2.2. Scaffold Technique

“A scaffold or matrix for a tissue engineering product refers to the ability to perform as a substrate that will support the appropriate cellular activity, including the facilitation of molecular and mechanical signalling systems, in order to optimize tissue regeneration, without eliciting any undesirable local or systemic response in the eventual host”. [44]
The scaffold technique is a tissue engineering technique, which uses three-dimensional structures made of hydrogels as a substrate to grow cells or tissues of interest. This method has gained immense popularity in the last few years and is widely used in IVM research [77][78] because scaffolds can act as a matrix for cell adherence to produce edible IVM skeletal muscle tissues. The scaffolds are made from hydrogels of natural or synthetic polymers, which are designed according to requirements and are later seeded with cells of interest. These cell-laden scaffolds are immersed in nutrient-rich medium contained in a bioreactor. Under favourable conditions, these cells grow into myotubes, which eventually form myofibrils. On maturation of cells, the resultant muscle fibres are harvested as edible IVM skeletal muscle tissue. Scaffolds developed using natural and edible hydrogels such as collagen and gelatin can produce complex meat with 3D structures. Thus, collagen and gelatin-based scaffolds are widely used to grow skeletal muscle tissues.

2.2.3. Bioreactors

The production of IVM can be carried out either using scaffolds or by scaffold-free techniques, such as the hanging drop method or agitation bioreactor method.
Bioreactors, are large enclosed stainless steel units used for culturing cells in a sterile manner, and they provide a favourable environment for the proliferation of cells [79][80]. The production of IVM is facilitated by biophysical factors such as agitation and shear that are achieved by the inclusion of bioreactors. Bioreactors are generally equipped with a media source, scaffolding system, oxygenation system and a plumbing system for the continuous inflow of media and outflow of metabolic wastes and recycled media. Here, the cells are either suspended freely or seeded onto a scaffold suspended in a bioreactor. The cells then undergo proliferation and differentiation to yield 3D muscle fibres, which can be potentially used as IVM. The bioreactor can help with IVM production in several ways. First, bioreactors help with the continuous suspension of culture media, so the cell culture is not deprived of the nutrient source. Second, it helps with agitating using a low shear so that the suspended tissues are unaffected. Third, it assists with adequate oxygen perfusion as oxygen gradient influences the cell viability and density. Fourth, it assists with continuous contraction of cells, which eventually undergoes differentiation to produce myofibers.
There are several types of bioreactors that can be used for IVM production. This includes the rotating wall/vessel [64][81], stir tank [82], direct perfusion [83], rotatory [84], hollow fibre [78][85], wave mixed [86], rotatory bed [31], parallel plate [87], fixed bed [88] and Synthecon [89] reactors. However, a stir tank bioreactor is the most used in IVM production [90][91][92][93].

2.2.4. Stimulation of Cells

In an in vivo system, all the cellular process occurs naturally. These processes are carried out by nerve stimulation and electrical transmission/stimulation, with the help of an extracellular matrix (ECM). However, this is not the case when cells are grown in vitro. The challenge lies in mimicking the in vitro environment like the in vivo environment. Thus, in vitro systems require external stimulation of cells, which is brought about in two ways, either by electrical or mechanical stimulation as described in the following sections.

Electrical Stimulation

The electrical stimulations mimic the nerve stimulations, which assist with the formation of highly differentiated and functional skeletal muscle tissue. Typically, electrical stimulation in in vitro is carried out by passing an electrical stimuli via salt bridges, which are dispensed in culture media [94]. There are a few setbacks with this method, such as a limited working area, making it difficult to work with various cell types at a time. Besides, the media-bridge system is susceptible to temperature fluctuations and exchange of salts and ions during electrical stimulation of cells can result in alteration of temperature, pH and salinity. Furthermore, the electrical stimulation system is incapable of running multiple chambers making it difficult to maintain sterility [42].
Electrical stimulation of cells during its growth is critical for IVM production for several reasons. First, electrical stimulation induces neuronal activity in the mature muscle fibres [95] and can be carried out by applying an electrical stimuli [96]. Second, electrical stimulation helps in accelerating the maturation of myotubules to develop early cross striations in C2C12 murine myoblasts. Third, electrical stimulation helps with neuronal activity by initiating contraction and differentiation of myotubules to eventually form myofibres [97].

Mechanical Stimulation

Mechanical stimulation is a biophysical stimulus that can be provided during myogenesis [47] as it influences gene expression, protein synthesis and total RNA/DNA content. Furthermore, it also helps with myofibre composition, cell number and muscle fibre diameter [40]. Mechanical stimulation of cells also helps with IVM production in several ways. First, it helps by applying mechanotransduction (a process through which cell sense and respond to mechanical stimuli by converting them to biochemical signals that elicit specific cellular responses), which alters the cell proliferation and differentiation rates [98]. Second, it helps with the fusion, alignment, and maturation of the myotube. Third, it helps with the proliferation and differentiation of muscle cells [99], muscle alignment [100] and muscle growth and maturation [101][102]. There are various methods of mechanical stimulation of cells [103]. The mechanical force is generated by using a perfusion bioreactor. When these mechanical forces are applied, this leads to perturbations in muscular protein conformation. This results in the exposure of hidden binding sites, which indirectly increases the signaling process in cells entrapped in the scaffold [104][105]. However, mechanical overloading can result in deformation, remodeling of cell, and can even affect cellular functions [106].

2.2.5. 3D Bioprinting

3D bioprinting is a novel method to create three-dimensional scaffolds of different hydrogel compositions. There are different types of printers available based on the technology, such as laser-assisted printing [106], as well as extrusion-based and inkjet-based 3D printing [105]. These 3D printers can efficiently create complex shapes of scaffolds with high resolution [107]. However, extrusion-based 3D printing is the most commonly used 3D printer [108]. The two main methods of 3D printing involve the use of either cellular scaffolds with cell-laden bio-ink or acellular scaffolds with hydrogels [109]. There are several ones on 3D printings for tissue engineering and regenerative medicine purposes [30][31][110][111][112], but not many on the 3D printing skeletal muscle cells for meat purposes [112][113].

3D Printing of Scaffolds

Extruded scaffolds can be developed using the Allevi 2 bioprinter. It is a Fused Deposition Modeling (FDM) bioprinter that runs on a compressed air pneumatic system [114]. It has two extruders, where the first extruder extrudes bio-inks, and the other extruder is used for photocuring (visible or UV light) extruded scaffolds. The hydrogel or bio-ink is dispensed in syringes with needles of 0.3 mm nozzle diameter to extrude scaffolds of synthetic polymers. The bioprinter works on a three-dimensional computer-aided design (CAD) software such as Slicr and Repertoire host, which helps in designing scaffolds. The 3D CAD models of the desired scaffolds are sliced into 2D cross-sections, to adjust printing parameters such as speed, in-fill density, gauges, nozzle diameter, print temperature, number of layers, layer height and air pressure. Software such as the Slicer or Repetier Host combines the two-dimensional cross sectionals of the scaffold to form a computer-aided three-dimensional structure. The bioprinter has a triaxial system (x, y, z), which allows scaffolds to be printed into desired shapes, which are then cured using built-in UV light to carry out crosslinking reactions.

Hydrogels

Hydrogels are three-dimensional polymer gels, which are made up of water-soluble polymers that are held together by water-insoluble cross-linkages. In other words, hydrogels are formed by the crosslinking of homopolymers or copolymers to give 3D structures with unique mechanical and chemical characteristics. Generally, most hydrogels swell and increase their weight when added to water due to their imbibition property. These hydrogels can be further classified into chemical or physical hydrogels based on the crosslinking mechanisms [37][115][116]. Physical hydrogels are not permanent, whereas chemical hydrogels are permanent. Hydrogels are either natural or synthetic in nature, and the natural hydrogels are comprised of polysaccharides and proteins and are usually found in the tissues such as agarose, gelatin, elastin, alginate, cellulose chitosan, fibrin, collagen and Matrigel [60][117] and hyaluronic acid. Natural hydrogels are more preferred due to their extracellular matrix-like structure, which enables cell growth, solute transport, cell binding and other cellular behaviours [37].
Synthetic hydrogels are usually made of polyethene glycol (PEG) [118], poly-(L-lactic acid) (PLLA) and polylactic-glycolic acid (PLGA) [119], polydimethylsiloxane (PDMS) [120], and poly(vinyl alcohol) (PVA) [114]. Semi-synthetic hydrogels such as gelatin methacrylate/gelatin methacryloyl (GelMA) have been used previously to culture cells [121]. Moreover, these synthetic hydrogels can be used in the production of highly structured 3D IVM because they can facilitate cell entrapment and cell growth. Hydrogels, in general, are extensively used in biomedical sciences applications [47][116][122][123] because they mimic extracellular matrix [124] and due to their biocompatibility, biodegradability, density and crosslinking properties. Moreover, these hydrogels offer a promising approach for skeletal muscle tissue engineering for many reasons. First, they allow dense cell entrapment uniformly in the hydrogel scaffold [125]. Second, they assist with myotube alignment due to the in vivo like environment. The major drawback with hydrogels is their instability, but this can be managed by co-culturing cells that produce extracellular matrix, which stabilises the matrix while the hydrogel degenerates [126]. The reproducibility and uniformity of the gels can be adjusted by electrospinning, but this may result in non-uniform distribution of cells.

Crosslinking Reaction

In chemistry, cross-links are referred to as bonds that connect one polymer chain to another through covalent bonds or ionic bonds. These cross-linkages are either formed by covalent [127][128] or noncovalent interactions [128][129] to form either chemical gels or physical gels, respectively. The process of crosslinking can be carried out either physically or chemically. Chemical crosslinking is carried out by either polymerization, chain-growth polymerization, sulphur vulcanization or by chemical reactions such as addition and condensation irradiation. It can also be performed by irradiation using high energy x-ray, electron beam and gamma rays. On the other hand, physical crosslinking is conducted by ionic interactions, crystallization, stereo complex formation, and protein interaction. Crosslinking is vital because it affects physicochemical properties of polymers such as elasticity, viscosity, swelling, solubility and strength of gels [130]. A detailed description of crosslinking of polymers is described in Ahadian et al. (2015) [116].

Polymerisation Reaction

Polymerisation is a crosslinking method where several monomer (homopolymers or copolymers) units react together chemically to form three dimensional polymers. Polymerisation is essential for hydrogels as it determines the physicochemical properties based on its monomers. A detailed description of the polymerisation reaction is beyond is described by Levental et al. (2009).

Types of Hydrogels

  • Collagen Hydrogel
Collagen is a naturally occurring protein that makes up 25% of the protein content in mammals [131]. Besides, it is the main component of extracellular matrix [132], which provides an in vivo-like environment that enables cell encapsulation, cell binding and integrin signalling [131]. In addition, collagen also provides exceptional crosslinking ability [116], low antigenicity, biodegradability [133] and higher biocompatibility [134]. Therefore, collagen hydrogels are widely used as a scaffold in tissue engineering [116]. However, they have a few drawbacks. First, they are soft and susceptible to degradation, but this can be managed either by increasing the amount of collagen or by chemically modifying it to prevent degradation [131]. Second, they can trigger an immune response occasionally, which can affect cell culture. Third, the usage of collagen hydrogels, in the long run, is not an economical option [135]. Finally, collagen demerits include thermal instability, low mechanical strength and susceptibility to contaminations [116][136].
  • Gelatin Hydrogels
Gelatin is a natural polymer, which is obtained by collagen hydrolysis. It is an economical, temperature-responsive polymer with high cell adhesiveness [137]. In addition, gelatin hydrogels are often functionalized with a cross-linkable component like methacryloyl group, which is crosslinked by photoinitiators [138] to enhance hydrogels stability [119]. However, most gelatin hydrogels are prone to hydrolysis, but this can be managed by chemical modification [139].
Gelatin methacryloyl (gelMA) is a modified form of gelatin, which is obtained by chemical crosslinking. It is one of the most widely used hydrogels because it offers excellent biocompatibility, physicochemical properties, printability and is cost effective [133][140]. Consequently, GelMA has been widely used in cell culture ones as an extracellular matrix due to its exceptional cell binding characteristics [141], as well as cell migration, differentiation and proliferation properties [142]. Nevertheless, there are a few demerits such as low mechanical strength [119][143][144], as well as reduced cell distribution and migration [145]. However, this can be managed by combining gelMA with hyaluronic acid or and silk fibroin [146].
  • Hyaluronic Acid Hydrogels
Hyaluronic acid (HA) or hyaluronan is a polysaccharide abundantly found in connective, epithelial and neural tissues. It is responsible for the formation of extracellular matrices with the help of glycosaminoglycans. Furthermore, it also helps with cell proliferation, cell migration, and other cellular functions. However, HA requires purification before hydrogel preparation to eliminate impurities and toxins. Hyaluronic acid has been used in many genetic engineering applications [144][145][146] because it is biocompatible with cells and mimics in vivo conditions. In addition, it helps with angiogenesis in engineered tissues [147] to promote vascularisation. Thus, HA has been widely used in the biomedical and tissue engineering research for over 30 years [147].

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